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Minivectors renew gene therapy hopes for lung treatments

Minivector™ DNA – very small circular therapeutic DNA molecules – survive the stress of aerosolization (being forced into suspension in air) and can carry gene therapy deep into the lungs, said researchers from Baylor College of Medicine and The University of Texas MD Anderson Cancer Center in a report that appears online in the journal Gene Therapy.

The ability to get deep in the lungs means that the Minivectors could be a potential treatment for a host of lung diseases, including lung cancer and asthma. These Minivectors, developed in the laboratory of Dr. Lynn Zechiedrich, associate professor of molecular virology and microbiology, biochemistry and molecular biology, and pharmacology at BCM, are not toxic, unlike existing vectors that are typically modified viruses. They survive longer than plasmids (large DNA circles containing bacterial sequences that can be turned off in human cells) and continue to work longer than small interfering RNAs, which are used now to turn off genes in the laboratory setting.

Minivectors show tremendous promise

"The ability of Minivectors to resist shear forces associated with gene therapy delivery renews hope of treating human diseases using non-viral DNA gene therapy vectors," said Zechiedrich.

"Minivectors show tremendous promise because of their tiny size," said Dr. Jamie Catanese, postdoctoral associate in the Zechiedrich laboratory and first author on the publication. "Because our lungs are accessible through the nose and mouth, lung diseases can be treated simply by breathing in the prescribed drug. However, the drug must be put into tiny droplets and this requires significant force."

One method of creating such droplets is the nebulizer, a machine that vaporizes liquid medicine into a fine mist. The mist is important in treating patients with breathing problems. Although this approach is routinely used to deliver drugs, previous attempts to use it with genetic material of the kind that would be used in gene therapy has resulted in the destruction of the DNA.

Zechiedrich and colleagues asked whether the tiny Minivectors, as short as 250 base pairs, could survive the process needed to make droplets small enough to get deep into the tiny branches of the lung, where DNA needs to go to treat disease. (The term base pairs refers to the coupling of two building blocks in DNA. T or thymidine always pairs to adenine. Cytosine always pairs with guanine.)

One worry was that the force of the nebulizer would tear DNA vectors apart and leave ends exposed. Those torn ends could attach to other pieces of DNA or even other chemicals in the cell to cause toxicity. Additionally, the ends might trigger mechanisms of DNA repair that could result in inflammation or even cell death.

To find out, they exposed the Minivectors to aerosolization, using a machine used in lung therapy. DNA destruction correlated strongly with its length. Typical plasmids, usually 3,000-plus base pairs, degraded quickly. Lengths of DNA from 2,000 to 3,000 base pairs survived about 10 minutes. Minivectors less than 1,200 base pairs completely survived the nebulization. Circular vectors survived much longer than linear ones of the same length. Supercoiling (coiling of the DNA about itself that makes the DNA even more compact) added additional protection from aerosolization.

Vectors must be circular, shorter

"Our data show that survival of shear forces associated with nebulization requires DNA vectors to be circular and shorter than 2,000 base pairs," Zechiedrich and colleagues wrote in the article.

Already this is long enough to bring in sequences that can deliver small genes or generate small RNAs to regulate how genes are turned on or off. Larger genes could be delivered by splitting them into smaller segments and then delivering these segments on multiple Minivectors in the body. The small segments then reconstitute into a functional protein.

"This is an old technology that we can use," said Catanese.

Others who took part in this research include Dr. Jonathan M. Fogg and Dr. Brian E. Gilbert of BCM, and Donald E. Schrock, II of MD Anderson.

Funding for this work came from the National Institutes of Health, the MD Anderson Molecular Genetic Technology Program and the Clayton Foundation for Research.